Chemical and Biological Engineering, Drexel University , Philadelphia, Pennsylvania 19104, United States.
Biochemistry. 2014 Jun 3;53(21):3403-14. doi: 10.1021/bi500136f. Epub 2014 May 19.
The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host-cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT-gp120 complex as a blueprint to design a covalently conjugated PT-gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21-E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT-gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT-gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT-gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT-gp120 conjugate to the noncovalent PT-gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT-gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced "off" state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.
HIV-1 gp120 糖蛋白是负责起始进入过程的主要病毒表面蛋白，因此可以作为开发进入抑制剂的靶标。我们之前鉴定了一类广泛有效的肽三唑（PT）双重拮抗剂，它们抑制 gp120 在其靶受体和共受体结合位点的相互作用，在病毒颗粒与宿主细胞接触之前诱导 gp120 的脱落，从而可以防止病毒进入和感染。然而，我们对 PT 引发其双重受体拮抗和病毒失活功能的 gp120 构象改变的理解是有限的。在这里，我们使用最近开发的 PT-gp120 复合物计算模型作为蓝图来设计一种共价连接的 PT-gp120 重组蛋白。最初，表达并表征了一个单一半胱氨酸 gp120 突变体，E275CYU-2。该变体对肽三唑、sCD4 和其他 CD4 结合位点（CD4bs）配体以及结合核心受体识别位点的 CD4 诱导（CD4i）配体保持极好的结合亲和力。同时，我们合成了一种 PEG 化和生物素化的肽三唑变体，该变体保留了 gp120 结合活性。该 PEG 化 PT 的 N 端马来酰亚胺变体，命名为 AE21，与 E275C gp120 缀合，生成 AE21-E275C 共价缀合物。表面等离子体共振相互作用分析表明，PT-gp120 缀合物显示出对 sCD4 和 17b 与 gp120 结合的抑制，这是包膜蛋白 PT 结合状态的特征。与非共价 PT-gp120 复合物类似，共价缀合物能够结合构象依赖性 mAb 2G12。结果表明，PT-gp120 缀合物是结构有序的，PT 和 gp120 结构域之间存在分子内相互作用，并且这种结构状态包含具有改变的桥接片但完整的 2G12 表位的构象捕获的 gp120。PT-gp120 缀合物与非共价 PT-gp120 复合物的相似性支持了在 PT-gp120 非共价复合物的分子动力学模拟模型中预测的 PT 与 gp120 结合的取向。构象稳定的共价缀合物可用于扩展 PT 诱导的 gp120“关闭”状态的结构定义，例如通过高分辨率结构分析。这种结构可以为提高基于结构的抑制剂的后续设计提供指导，这些抑制剂具有肽三唑作用模式。
